The most commonly used method for assessing the penetration of agents (e.g. drugs and pharmaceuticals) into living tissue is the multi-cellular spheroid. Multicellular spheroids are a conglomeration of up to 10.sup.6 animal cells which form small balls of tissue and are grown in a flask in which the media is stirred (spinner culture). Penetration of agents into such multi-cellular spheroids can be monitored by applying the agent for a period of time, then removing spheroids, sectioning and then microscopically examining them.
The use of the spheroid system the measuring penetration is effective, but has several significant limitations. The geometry of the diffusion or transport into spheroids is almost inverse of some situations encountered in nature, i.e. in many instances drugs diffuse radially outward from capillaries within tissue, while with the spheroids the agent diffuses from the outer or maximum surface area toward the center of the sphere. The main drawback of using spheroids to measure penetration of an agent is the requirement that the concentrations of the agent at different depths of penetration cannot be determined easily. Usually after a period of time has lapsed to allow penetration of the test agent the spheroids are removed from the incubating medium and quickly frozen, so they may then be section and evaluated. When the agent is fluorescent it is microscopically evaluated. If radio labeled (which is expensive and more difficult) a photographic emulsion is applied and then the section microscopically evaluated. The agent must bind to the cells to permit dissociation from the spheroid and analysis using flow cytometry. If the agents are not readily identifiable (i.e. colored, fluorescent or radio labeled) spheroids cannot be used.
In some other conventional techniques for producing cells, the cells are grown as a single cell layer on a plastic or glass surface e.g. in a petri dish and have one face directly facing the petri dish so that substantially all the growth occurs from the opposite face. Cells stop growing when they reach confluence or form a super-confluence and become senescent due to oxygen/nutrient deprivation.
U.S. Pat. No. 5,160,490 issued Nov. 3, 1991 to Naughton et al. describes a three-dimensional cell culture system for culturing a variety of different cells and tissues in-vitro for extended periods of time by growing same on a pre-established stroma cell matrix.
In an article entitled "Three-Dimensional Histoculturez Origins and Applications in Cancer Research" by Hoffman published in Cancer Cells, March 1991, Volume 3 Number 3 a review of known techniques for growing three dimensional histocultures is presented and describes sponge-matrix cultures, Collagen-gel cultures, filter or mesh supported organ cultures and spheroids. The paper also reviews some studies of tumor cell migrations and invasion and drag sensitivity assays. None of these techniques is available for growing cell cultures in-vitro wherein the cell culture grown is at least 5 cells in thickness and wherein the so formed culture provides a relatively uniform thickness mat of cells which may then be used to investigate the effectiveness of applied reagents.
Currently, anti-cancer agents are mainly evaluated experimentally using in-vitro methods (cells growing in mono layers or in suspension cultures or spheroids) and in-vivo systems utilizing animals, i.e. to determine their activity in-vitro against cells in the mono layer culture. When an active agent is identified, the compound is transferred to in-vivo testing systems wherein it is administered to animals and the effect against tumors such as implated tumors is assessed. This transfer from in-vitro to in-vivo testing is generally quite problematic in that the animal physiology and the process of drug absorption, distribution, and the metabolism of the animal and various excretions often alter the behavior of the drugs from that predicted by the in-vitro studies.
U.S. Pat. No. 4,667,504 issued May 26, 1987 to Hobson; 5,183,760 issued Feb. 2, 1993 to Sweetana et al. and 5,198,109 issued Mar. 30, 1993 to Hanson et al. each disclose diffusion cells as means for testing the diffusion of chemicals through a membrane of cells. Similar systems to those disclosed in the patents are sold by Corning Costar corporation under the trademark COSTAR.
One of the major problems that is encountered in administering drugs to counteract a tumor growth, is the requirement of the drug to penetrate the tumor and there are few means available to assess such penetration.